The development of the symbiosis that results in nodule formation and nitrogen fixation is a complex process that involves genetic factors in both the bacterial symbiont and the host plant. Much work has been done to elucidate the genetic factors in Rhizobium that affect symbiosis. In contrast, only a few heritable host plant factors have been reported to influence the extent of symbiosis in legumes (Nutman (1981) in Current Perspectives in Nitrogen Fixation Research, Gibson and Newton (eds) Austr. Acad. Sci., Canberra, Australia, pp. 194-204). The most striking host variations in nodulation that have been observed are decreased nodulation or absence of nodulation. However, natural variants within existing germ plasms with enhanced nodulation have been demonstrated in some species of legumes (Gelin and Blixt (1964) Agri Hort. Genet. 22:149-163; Nutman (1953) Heredity 8:35-46; Duhigg et al. (1978) Crop Sci. 18:813-816; Heichel et al. (1984) in Advances in Nitrogen Fixation Research, Veeger and Newton (eds) Martinus Nijhof/Junk, The Hague Netherlands, p. 595; Imsande (1984) ibid. p. 596; Nutman et al. (1971) in Biological Nitrogen Fixation in Natural and Agricultural Habitats, Lie and Mulder (eds) (Plant and Soil special volume) Martinus Nijhoff, The Hague, Netherlands pp. 27-31; Mylton and Jones (1971) ibid. pp. 17-25).
Symbiotic nitrogen fixation in legumes can also be affected by environmental factors. One such factor that is particularly important is the level of nitrate in the soil. The development of symbiosis is inhibited by high soil nitrate concentrations. Suppression of symbiosis is manifested both in reduced nodule mass and in reduced nitrogenase activity per unit nodule mass (Carroll, B. J. and Gresshoff, P. M. (1983) Z. Pflanzenphysiol. 100:77-88; Streeter, J. G. (1981) Plant Physiol. 68:840-844). The mechanism of nitrate inhibition is not fully understood, but it is hypothesized either that nitrate reduction which limits carbohydrate availability results in limitation of nodule development or that the products of nitrate reduction, particularly nitrite, inhibit nitrogenase (Oghoghorie and Plate (1971) in Biological Nitrogen Fixation in Natural and Agricultural Habitats, Plant and Soil Special Volume, Lie and Mulder (eds) Nijhoff/Junk, The Hague, The Netherlands, p. 185-202; Trinchant and Rigaud (1980) Arch. Microbiol. 124:49-54; Rigaud and Puppo (1977) Biochim. Biophys. Acta 497:702-706). Minor differences in nodulation tolerance to nitrate have been demonstrated between host species (Carroll et al. (1984) in Advances in Nitrogen Fixation Research, Veeger and Newton (eds) Nijhoff/Junk, The Hague, The Netherlands, p. 589) and between cultivars within a species (Carroll et al., 1984; Harper and Gibson (1984) in Advances in Nitrogen Fixation Research, Veeger and Newton (eds) Nijhoff/Junk, The Hague, The Netherlands, p. 589) suggesting that host genetic factors affect nitrate-tolerance of nodulation.
Recently, Jacobsen (1984) in Advances in Nitrogen Fixation Research, Veeger and Newton (eds) Nijhoff/Junk, The Hague, The Netherlands, p. 597 and Jacobsen and Feenstra (1984) Plant Science Letters 33:337-344 reported the generation by mutagenesis and selection of one pea mutant that nodulates on nitrate containing medium. The mutation was described as monogenic and recessive.
It had previously been reported that extent of nodulation in legumes was affected by legume genetic factors. Gelin and Blixt, 1964, reported that the considerable variation in nodule number among pea lines that they observed was genetically controlled and that two genes (then designated No and Nod, now nod1 and nod2) were involved. Nitrate-tolerant nodulation was not reported to be associated with these genetic factors.
Although it has recently been reported (Carroll et al., 1984) that there is some variation in sensitivity of soybean cultivars to nitrate, nitrate-tolerance of nodulation has not been observed in populations of wild or domestic cultivars of soybean. Prior to the present invention, it was not known or understood that genetic manipulation of a plant could achieve a plant phenotype of nitrate-tolerant nodulation, since both plant and bacterial genes are involved in establishing effective root nodules and genetic studies involving genetic manipulation of the symbiosis has been confined to Rhizobium.
Some of the results described herein, have been reported in Carroll et al. (1985) Proc. Nat'l. Acad. Sci. U.S.A. 82:4162-4166; and Carroll et al. (1985) Plant Physiol. 78:34-40.